Identification of mhc class i phospho-peptide antigens from breast cancer utilizing shla technology and complementary enrichment strategies

ABSTRACT

The present invention describes novel tumor-specific phosphorylated peptides, nucleic acids encoding those peptides, and antibodies generated against said peptides. The genes, peptides, and antibodies described herein may be used as diagnostic indicators of the presence of breast cancer and/or used in therapeutics to treat breast cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/403,350, filed Mar. 12, 2015, which is a 35 U.S.C. § 371 filing ofInternational Patent Application No. PCT/US2013/042908, filed May 28,2013, which claims priority to U.S. Provisional Patent ApplicationSerial Nos. 61/652,028, filed May 25, 2012; and 61/667,697, filed Jul.3, 2012, the disclosures of each of which are incorporated by referenceherein in their entireties.

U.S. GOVERNMENT RIGHTS

This invention was made with United States Government support underGrant No. R01 AI33993 awarded by the National Institutes of Health. TheUnited States Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 2, 2020, isnamed 702696_AGBW-075USCON_ST25.txt and is 39,295 bytes in size.

BACKGROUND

The mammalian immune system has evolved a variety of mechanisms toprotect the host from cancerous cells. An important component of thisresponse is mediated by cells referred to as T cells. Cytotoxic Tlymphocytes (CTL) are specialized T cells that primarily function byrecognizing and killing cancerous cells or infected cells, but they canalso function by secreting soluble molecules referred to as ytokinesthat can mediate a variety of effects on the immune system. T helpercells primarily function by recognizing antigen on specialized antigenpresenting cells, and in turn secreting cytokines that activate B cells,T cells, and macrophages.

A variety of evidence suggests that immunotherapy designed to stimulatea tumor-specific CTL response would be effective in controlling cancer.For example, it has been shown that human CTL recognize sarcomas (Slovinet al., 1986, J Immunol 137, 3042-3048), renal cell carcinomas (Schendelet al., 1993, J Immunol 151, 42094220), colorectal carcinomas (Jacob etal., 1997, Int J Cancer 71, 325-332), ovarian carcinomas (Peoples etal., 1993, Surgery 114, 227-234), pancreatic carcinomas (Peiper et al.,1997, Eur J Immunol 27, 1115-1123), squamous tumors of the head and neck(Yasumura et al., 1993, Cancer Res 53, 1461-1468), and squamouscarcinomas of the lung (Slingluff et al., 1994, Cancer Res 54,2731-2737; Yoshino et al., 1994, Cancer Res 54, 3387-3390). The largestnumber of reports of human tumor-reactive CTLs, however, has concernedmelanomas (Boon et al., 1994, Annu Rev Immunol 12, 337365). The abilityof tumor-specific CTL to mediate tumor regression, in both human(Parmiani et al., 2002, J Natl Cancer Inst 94, 805-818; Weber, 2002,Cancer Invest 20, 208-221) and animal models, suggests that methodsdirected at increasing CTL activity would likely have a beneficialeffect with respect to tumor treatment.

Melanoma, or skin cancer, is a disease that is diagnosed inapproximately 54,200 persons per year. Conventional therapy for thedisease includes surgery, radiation therapy, and chemotherapy. In spiteof these approaches to treatment, approximately 7,600 individuals die inthe United States every year due to melanoma. Overall, the 5-yearsurvival rate for the disease is 88%. The survival rate drops, however,in more advanced stages of the disease with only about 50% of Stage IIIpatients, and 20-30% of Stage IV patients surviving past five years. Inpatients where the melanoma has metastasized to distant sites, the5-year survival dips to only 12%. Clearly, there is a population ofmelanoma patients that is in need of better treatment options. Morerecently, in an attempt to decrease the number of deaths attributed tomelanoma, immunotherapy has been added to the arsenal of treatments usedagainst the disease.

In order for CTL to kill or secrete cytokines in response to a cancercell, the CTL must first recognize the cancer cell (Townsend and Bodmer,1989). This process involves the interaction of the T cell receptor,located on the surface of the CTL, with what is generically referred toas an MHC-peptide complex which is located on the surface of thecancerous cell. MHC (major histocompatibility-complex)-encoded moleculeshave been subdivided into two types, and are referred to as class I andclass II MHC-encoded molecules. In the human immune system, MHCmolecules are referred to as human leukocyte antigens (HLA). Within theMHC complex, located on chromosome six, are three different loci thatencode for class I MEW molecules. MEW molecules encoded at these lociare referred to as HLA-A, HLA-B, and HLA-C. The genes that can beencoded at each of these loci are extremely polymorphic, and thus,different individuals within the population express different class IMEW molecules on the surface of their cells. HLA-A1, HLA-A2, HLA-A3,HLA-B7, and HLA-B8 are examples of different class I MHC molecules thatcan be expressed from these loci.

The peptides which associate with the MHC molecules can either bederived from proteins made within the cell, in which case they typicallyassociate with class I MHC molecules (Rock and Goldberg, 1999, Annu RevImmunol 17, 739-779); or they can be derived from proteins which areacquired from outside of the cell, in which case they typicallyassociate with class II MHC molecules (Watts, 1997, Annu Rev Immunol 15,821-850). The peptides that evoke a cancer-specific CTL response mosttypically associate with class I MHC molecules. The peptides themselvesare typically nine amino acids in length, but can vary from a minimumlength of eight amino acids to a maximum of twelve amino acids inlength. Tumor antigens may also bind to class II MEW molecules onantigen presenting cells and provoke a T helper cell response. Thepeptides that bind to class II MHC molecules are generally twelve tonineteen amino acids in length, but can be as short as ten amino acidsand as long as thirty amino acids.

The process by which intact proteins are degraded into peptides isreferred to as antigen processing. Two major pathways of antigenprocessing occur within cells (Rock and Goldberg, 1999, Annu Rev Immunol17, 739-779). One pathway, which is largely restricted to cells that areantigen presenting cells such as dendritic cells, macrophages, and Bcells, degrades proteins that are typically phagocytosed or endocytosedinto the cell. Peptides derived in this pathway typically bind to classII MEW molecules. A second pathway of antigen processing is present inessentially all cells of the body. This second pathway primarilydegrades proteins that are made within the cells, and the peptidesderived from this pathway primarily bind to class I MEW molecules.Antigen processing by this latter pathway involves polypeptide synthesisand proteolysis in the cytoplasm, followed by transport of peptides tothe plasma membrane for presentation. These peptides, initially beingtransported into the endoplasmic reticulum of the cell, becomeassociated with newly synthesized class I MHC molecules and theresulting complexes are then transported to the cell surface. Peptidesderived from membrane and secreted proteins have also been identified.In some cases these peptides correspond to the signal sequence of theproteins which is cleaved from the protein by the signal peptidase. Inother cases, it is thought that some fraction of the membrane andsecreted proteins are transported from the endoplasmic reticulum intothe cytoplasm where processing subsequently occurs.

Once bound to the class I MHC molecule, the peptides are recognized byantigen-specific receptors on CTL. Several methods have been developedto identify the peptides recognized by CTL, each method of which relieson the ability of a CTL to recognize and kill only those cellsexpressing the appropriate class I MHC molecule with the peptide boundto it. Mere expression of the class I MHC molecule is insufficient totrigger the CTL to kill the target cell if the antigenic peptide is notbound to the class I MHC molecule. Such peptides can be derived from anon-self source, such as a pathogen (for example, following theinfection of a cell by a bacterium or a virus) or from a self-derivedprotein within a cell, such as a cancerous cell. The tumor antigens fromwhich the peptides are derived can broadly be categorized asdifferentiation antigens, cancer/testis antigens, mutated gene products,widely expressed proteins, and viral antigens (Castelli et al., 2000, JCell Physiol 182, 323-331).

Immunization with melanoma-derived, class I or class II MHC-encodedmolecule associated peptides, or with a precursor polypeptide or proteinthat contains the peptide, or with a gene that encodes a polypeptide orprotein containing the peptide, are forms of immunotherapy that can beemployed in the treatment of melanoma. This form of immunotherapyrequires that immunogens be identified so that they can be formulatedinto an appropriate vaccine. Although a large number of tumor-associatedpeptide antigens recognized by tumor reactive CTL have been identified,there are few examples of antigens that are derived from proteins thatare selectively expressed on a broad array of tumors, as well asassociated with cellular proliferation and/or transformation. Attractivecandidates for this type of antigen are peptides derived from proteinsthat are differentially phosphorylated on serine (Ser), threonine (Thr),and tyrosine (Tyr) (Zarling et al., 2000, J Exp Med 192 1755-1762). Dueto the increased phosphorylation of cellular proteins in transformedcells as compared to normal cells, there are likely to be newphosphorylated peptides presented on the cell surface available forrecognition by CTL. However, these are not predictable from simpleinspection of protein sequences, and the exact phosphorylation sites ofmany proteins, as well as their phosphorylation state in a tumor cell,remain unknown.

There is a long felt need in the art for methods of identifying tumorantigens, and for methods of treating or preventing cancer based on theuse of such tumor antigens. The present invention satisfies these needs.

SUMMARY OF THE INVENTION

The present invention describes novel tumor-specific peptides andantibodies generated against said peptides. The peptides and antibodiesdescribed herein may be used as diagnostic indicators of the presence ofcancer and/or used in therapeutics to treat and prevent cancer. For thepresent invention, mass spectrometry has been used to identifyphosphorylated peptides associated with the class I WIC moleculeHLA-A*0201 and HLA-B7 and displayed on melanoma cells. The inventionalso provides novel methods for identifying such peptides.

In one aspect, the cancer is breast cancer.

Various aspects and embodiments of the invention are described infurther detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 , comprising FIGS. 1A and 1B, is a graphic illustration of therecognition of naturally processed and presented phosphorylated peptideson cancer cells by the phosphopeptide-specific CTL.Phosphopeptide-specific CTL were incubated with the following cancercell lines or EBV-transformed B lymphoblastoid cell lines (BLCL):COV413.AAD.A4 ovarian carcinoma, DM331.AAD.A4 and SLM2.AAD.A1 melanomas,MCF7.AAD.A2 and MDAMB231.AAD breast carcinomas, and JY EBV-BLCL.Supernatants were harvested and evaluated for the presence of murineIFNγ (produced by murine CTL lines). As a positive control, cancer cellswere pulsed with the specific phosphopeptide to show that they arecapable of presenting exogenously added peptide. In FIG. 1A, twophosphopeptide-specific CTL cell lines, 6850 and 6960 that are specificfor the phosphopeptide GLLGpSPVRA, recognize the phosphopeptide on allthe cancer cell lines, but not the control cell line. In FIG. 1B, twophosphopeptide-specific CTL cell lines, 5183 and 63 that are specificfor the phosphopeptide RVApSPTSGV, recognize the phosphopeptide on allthe cancer cell lines, but not the control cell line. The ordinateindicates murine IFNγ in pg/ml. The abscissa indicates each cell line.

FIG. 2 presents a Table of phosphopeptides identified for breast cancerby the Class I MHC Molecule on the HLA B*0702 allele.

FIG. 3 graphically demonstrates the MHC class I pathway.

FIG. 4 schematically illustrates an overview of sample analysis.

FIG. 5 describes the B*0702 motif showing amino acid anchors.

FIG. 6 shows the total number of peptides in IMAC and TiO2 experimentsand schematically depicts the overlap between the peptides identified inan IMAC analysis of 184B5 and a TiO2 analysis.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, the articles “a” and “an” refer to one or to more thanone, i.e., to at least one, of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

The expression “amino acid” as used herein is meant to include bothnatural and synthetic amino acids, and both D and L amino acids.“Standard amino acid” means any of the twenty standard L-amino acidscommonly found in naturally occurring peptides. “Nonstandard amino acidresidue” means any amino acid, other than the standard amino acids,regardless of whether it is prepared synthetically or derived from anatural source. As used herein, “synthetic amino acid” also encompasseschemically modified amino acids, including but not limited to salts,amino acid derivatives (such as amides), and substitutions. Amino acidscontained within the peptides of the present invention, and particularlyat the carboxy- or amino-terminus, can be modified by methylation,amidation, acetylation or substitution with other chemical groups whichcan change the peptide's circulating half-life without adverselyaffecting their activity. Additionally, a disulfide linkage may bepresent or absent in the peptides of the invention.

The term “amino acid” is used interchangeably with “amino acid residue,”and may refer to a free amino acid and to an amino acid residue of apeptide. It will be apparent from the context in which the term is usedwhether it refers to a free amino acid or a residue of a peptide.

Amino acids have the following general structure:

Amino acids may be classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup. The nomenclature used to describe the peptide compounds of thepresent invention follows the conventional practice wherein the aminogroup is presented to the left and the carboxy group to the right ofeach amino acid residue. In the formulae representing selected specificembodiments of the present invention, the amino- and carboxy-terminalgroups, although not specifically shown, will be understood to be in theform they would assume at physiologic pH values, unless otherwisespecified.

The term “basic” or “positively charged” amino acid as used herein,refers to amino acids in which the R groups have a net positive chargeat pH 7.0, and include, but are not limited to, the standard amino acidslysine, arginine, and histidine.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, are reduced. The term “antigen” asused herein is defined as a molecule that provokes an immune response.This immune response may involve either antibody production, or theactivation of specific immunologically-competent cells, or both.

As used herein, the term “antibody” refers to a polyclonal or monoclonalantibody or a binding fragment thereof such as Fab, F(ab′)2 and Fvfragments.

As used herein, the term “antigen peptide” refers to a phosphorylatedamino acid sequence derived from a cancer cell, such as the sequencesselected from the group consisting of SEQ ID NOs:70-171 or a peptide atleast or about 80, 85, 90, 95, 98, 99, or 100% identical thereto.

As used herein, the term “cancer cell-specific phosphopeptide” refers toa phosphopeptide, which is expressed at higher levels in said cancercell compared to its normal counterpart cell or tissue.

A “control” cell, tissue, sample, or subject is a cell, tissue, sample,or subject of the same type as a test cell, tissue, sample, or subject.The control may, for example, be examined at precisely or nearly thesame time the test cell, tissue, sample, or subject is examined. Thecontrol may also, for example, be examined at a time distant from thetime at which the test cell, tissue, sample, or subject is examined, andthe results of the examination of the control ay be recorded so that therecorded results may be compared with results obtained by examination ofa test cell, tissue, sample, or subject.

A “test” cell, tissue, sample, or subject is one being examined.

A “pathoindicative” cell, tissue, or sample is one which, when present,is an indication that the animal in which the cell, tissue, or sample islocated (or from which the tissue was obtained) is afflicted with adisease or disorder. By way of example, the presence of one or morebreast cells in a lung tissue of an animal is an indication that theanimal is afflicted with metastatic breast cancer.

A tissue “normally comprises” a cell if one or more of the cell arepresent in the tissue in an animal not afflicted with a disease ordisorder.

As used herein, a “functional” biological molecule is a biologicalmolecule in a form in which it exhibits a property or activity by whichit is characterized. A functional enzyme, for example, is one whichexhibits the characteristic catalytic activity by which the enzyme ischaracterized. “Homologous” as used herein, refers to the subunitsequence similarity between two polymeric molecules, e.g., between twonucleic acid molecules, e.g., two DNA molecules or two RNA molecules, orbetween two polypeptide molecules. When a subunit position in both ofthe two molecules is occupied by the same monomeric subunit, e.g., if aposition in each of two DNA molecules is occupied by adenine, then theyare homologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′ATTGCC5′ and 3′TATGGC share 50%homology.

As used herein, “homology” is used synonymously with “identity.”

The determination of percent identity between two nucleotide or aminoacid sequences can be accomplished using a mathematical algorithm. Forexample, a mathematical algorithm useful for comparing two sequences isthe algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl.Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into theNBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol.215:403-410), and can be accessed, for example at the National Centerfor Biotechnology Information (NCBI) world wide web site. BLASTnucleotide searches can be performed with the NBLAST program (designated“blastn” at the NCBI web site), using the following parameters: gappenalty=5; gap extension penalty=2; mismatch penalty=3; match reward=1;expectation value 10.0; and word size=11 to obtain nucleotide sequenceshomologous to a nucleic acid described herein. BLAST protein searchescan be performed with the XBLAST program (designated “blastn” at theNCBI web site) or the NCBI “blastp” program, using the followingparameters: expectation value 10.0, BLOSUM62 scoring matrix to obtainamino acid sequences homologous to a protein molecule described herein.To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997, Nucleic Acids Res.25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used toperform an iterated search which detects distant relationships betweenmolecules (Id.) and relationships between molecules which share a commonpattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

By the term “immunizing a subject against an antigen” is meantadministering to a subject a composition, a peptide, a polypeptide, or afragment, derivative, or modification thereof, a protein complex, a DNAencoding a protein complex, an antibody or a DNA encoding an antibody,which elicits an immune response in the human which immune responseprovides protection to the human against a disease caused by the antigenor an organism which expresses the antigen.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression, which can beused to communicate the usefulness of the composition of the inventionfor its designated use. The instructional material of the kit of theinvention may, for example, be affixed to a container, which containsthe composition or be shipped together with a container which containsthe composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the composition be used cooperatively by therecipient.

As used herein, a “ligand” is a compound that specifically binds to atarget compound or molecule. A ligand “specifically binds to” or “isspecifically reactive with” a compound when the ligand functions in abinding reaction which is determinative of the presence of the compoundin a sample of heterogeneous compounds.

As used herein, a “peptide” encompasses a sequence of 3 or more aminoacids wherein the amino acids are naturally occurring or synthetic(non-naturally occurring) amino acids. Peptide mimetics include peptideshaving one or more of the following modifications:

-   -   1. peptides wherein one or more of the peptidyl —C(O)NR—        linkages (bonds) have been replaced by a non-peptidyl linkage        such as a —CH2-carbamate linkage (—CH₂OC(O)NR—), a phosphonate        linkage, a —CH2-sulfonamide (—CH2-S(O)2NR—) linkage, a urea        (—NHC(O)NH—) linkage, a —CH2-secondary amine linkage, or with an        alkylated peptidyl linkage (—C(O)NR—) wherein R is C1-C4 alkyl;    -   2. peptides wherein the N-terminus is derivatized to a —NRR₁        group, to a —NRC(O)R group, to a —NRC(O)OR group, to a —NRS(O)2R        group, to a —NHC(O)NHR group where R and R₁ are hydrogen or        C1-C4 alkyl with the proviso that R and R1 are not both        hydrogen;    -   3. peptides wherein the C terminus is derivatized to —C(O)R2        where R2 is selected from the group consisting of C1-C4 alkoxy,        and —NR3R4 where R3 and R4 are independently selected from the        group consisting of hydrogen and C1-C4 alkyl.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

The term “protein” typically refers to large polypeptides.

Naturally occurring amino acid residues in peptides are abbreviated asrecommended by the IUPAC-IUB Biochemical Nomenclature Commission asfollows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine isIle or I; Methionine is Met or M; Norleucine is Nle; Valine is Val or V;Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanineis Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine isGln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid isAsp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan isTrp or W; Arginine is Arg or R; Glycine is Gly or G, and Xaa or X is anyamino acid. Other naturally occurring amino acids include, by way ofexample, 4-hydroxyproline, 5-hydroxylysine, and the like.

Synthetic or non-naturally occurring amino acids refer to amino acidswhich do not naturally occur in vivo but which, nevertheless, can beincorporated into the peptide structures described herein. The resulting“synthetic peptide” contains amino acids other than the 20 naturallyoccurring, genetically encoded amino acids at one, two, or morepositions of the peptides. For instance, naphthylalanine can besubstituted for tryptophan to facilitate synthesis. Other syntheticamino acids that can be substituted into peptides includeL-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, alpha-amino acids such asL-alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha.-methylalanyl,beta.-amino acids, and isoquinolyl. D amino acids and non-naturallyoccurring synthetic amino acids can also be incorporated into thepeptides. Other derivatives include replacement of the naturallyoccurring side chains of the 20 genetically encoded amino acids (or anyL or D amino acid) with other side chains.

As used herein, the term “conservative amino acid substitution” isdefined herein as an amino acid exchange within one of the followingfive groups:

I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr,Pro, Gly;

IL Polar, negatively charged residues and their amides: Asp, Asn, Glu,Gln;

III. Polar, positively charged residues: His, Arg, Lys;

IV. Large, aliphatic, nonpolar residues: Met Leu, Ile, Val, Cys

V. Large, aromatic residues: Phe, Tyr, Trp

As used herein, the term “purified” and like terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

A “subject” of diagnosis or treatment is a mammal, including a human.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms. A “prophylactic” treatment is a treatment administered to asubject who does not exhibit signs of a disease or exhibits only earlysigns of the disease for the purpose of decreasing the risk ofdeveloping pathology associated with the disease.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

A “therapeutically effective amount” of a compound is that amount ofcompound which is sufficient to provide a beneficial effect to thesubject to which the compound is administered.

By the term “vaccine,” as used herein, is meant a composition, whichwhen inoculated into a mammal has the effect of stimulating a cellularimmune response comprising a T cell response or a humoral immuneresponse comprising a B cell response generally resulting in antibodyproduction. The T cell response may be a cytotoxic T cell responsedirected against macromolecules produced by the bacteria. However, theinduction of a T cell response comprising other types of T cells by thevaccine of the invention is also contemplated. A B cell response resultsin the production of antibody which binds to the antigen. The “vaccine”has the effect of stimulating an immune response in the subject, whichserves to fully or partially protect the subject against a disease orits symptoms. The term vaccine encompasses prophylactic as well astherapeutic vaccines. A combination vaccine is one which combines two ormore vaccines.

EMBODIMENTS OF THE INVENTION

The present invention is directed to novel phosphorylated peptides thatgive rise to cancer antigens. In one embodiment, for example, thephosphopeptides are those described in the tables of the invention,including Table 1, associated with Examples 1 and 2. Example 3 andAppendix have different Tables with other peptides, and all 5 sequencescited below are for Table 1 of Examples 1 and 2, but the embodimentscited are also useful for the results and sequences of Example 3 andAppendix A. In one aspect, the peptides bind to MHC molecules. Inanother aspect, the peptides of the invention stimulate an immuneresponse. In yet another aspect, the peptides of the invention arerecognized by a cell or molecule which is a product of, or is stimulatedas 10 a result of, an immune response.

TABLE 1 Cancer Antigen Phosphopeptides Gi Protein number SequenceHLA type SEQ ID MUM-2 20177848 RLDpSYVRSL HLA-A2.1  1Orphan nuclear receptor T2   136117 RQDpSTPGKVFL HLA-A2.1  2Riken ORF 32, chromosome 10 58864795 VLKGpSRSSEL HLA-A2.1  3ORF 17, chromosome 2 40787650 RLpSSPLHFV HLA-A2.1  4ATP-dependent metalloprotease 14248493 RLQpSTSERL HLA-A2.1  5Heterogeneous nuclear 13938287 AMAApSPHAV HLA-A2.1  6ribonucleoprotein: AO Jun-b/c/d 49456463 KLApSPELERL HLA-A2.1  7Ribosomal protein L4 22002063 ILKpSPEIQRA HLA-A2.1  8Ub-carboxyl terminal  2501458 KLLpSPSNEKL HLA-A2.1  9hydrolase-10 (USP-10) Ribosomal protein S17 51476007 KLLDFGSLpSNLQVHLA-A2.1 10 Krueppel-like zinc finger protein   903598 KLLSSAQRpTLHLA-A2.1 11 B-Catenin 20384898 YLDpSGIHSGA HLA-A2.1 12 CDC25b: p6314602917 GLLGpSPVRA HLA-A2.1 13 Insulin receptor substrate-2 55663292RVApSPTSGV HLA-A2.1 14 Breast cancer anti-estrogen 55663999 IMDRpTPEKLHLA-A2.1 15 resistance-3 (BCAR3) Tumor endothelial marker-6, 23451123VMIGpSPKKV HLA-A2.1 16 thyroid specific PTB domain proteinHypothetical protein FAM65A 32493393 RTLpSHISEA HLA-A2.1 17 proteinNedd4 binding protein 2, 31742492 KMDpSFLDMQL HLA-A2.1 18BCL3 binding protein Unknown (protein gi: 22902182) 22902182LMFpSVTS(L/I) HLA-A2.1 19 Pleckstrin homology 46397654 SLQPRSHpSVHLA-A2.1 20 domain-containing protein family A member 6Predicted: similar to RAVER1 55648233 RLLpSPLSSA HLA-A2.1 21SRp46 splicing factor 14141201 SMpTRSPPRV HLA-A2.1 22Adenosine monophosphate 56206061 RQIpSQDVKL HLA-A2.1 23deaminase 2 (isoform L) B lymphocyte signal  4261606 RQApSIELPSMHLA-A2.1 24 transduction gene B lymphocyte signal  4261606 RQApSIELPSMAVHLA-A2.1 25 transduction gene Carcinoembryonic antigen 2b  3702267SLLTFWNL HLA-A2.1 26 SLTP004 20146522 KVQVpTSLSV HLA-A2.1 27Tsg24 protein 11967711 VLLpSPVPEL HLA-A2.1 28 YME1-like 1, isoform 214043646 RLQpSTSERL HLA-A2.1 29 FLJ22624 protein 55661789 TLApSPSVFKSTHLA-A2.1 30 Premature ovarian failure, 1B 57284143 RTYpSGPMNKV HLA-A2.131 Serine/threonineprotein  7531055 KLIDIVpSSQKV HLA-A2.1 32kinase Chk1. Desmuslin, synemin 20137613 RTFpSPTYGL HLA-A2.1 33FLJ20297 protein, 40674045 ALYpSPAQPSL HLA-A2.1 34 KIAA1418 proteinAdenosine monophosphate 56206061 RQIpSQDVKL HLA-A2.1 35deaminase 2 (isoform L) BRD4_Human bromodomain 20141192 AVVpSPPALHNAHLA-A2.1 36 containing protein 4 MUM-2 20177848 RLDpSYVRS HLA-A2.1 37Pro-apoptotic HPKRSVpSL HLA-B7 38 protein/BCL2adenovirusE1B interacting protein 31ike/My020 proteinKIAA1187/FLJ10350/Hypothetical LPApSPRARL HLA-B7 39 protein FLJ11029HPRpSPTPTL HLA-B7 40 protein/unnamed protein signal-induced YPSpSPRKALHLA-B7 41 proliferation-associated 1 like 1/high-risk humanpapilloma viruses E6 oncoproteins targeted protein Paternally expressedKPRpSPPRAL HLA-B7 42 gene 10 ORF1 Novel protein/similar to cdc42RPAKpSMDSL HLA-B7 43 GTPase activating protein Beta-adrenergi c-receptorKPRpSPVVEL HLA-B7 44 kinase No direct database hit- (X = L/I)Suppressor of cytokine APRpSPPPSRP HLA-B7 46 signaling proteininositol 1,4,5-triphosphate RPSGRREpSL HLA-B7 47 receptor, type 1Tumor necrosis factor RPRRpSSTQL HLA-B7 48 receptor superfamily,member 8 (CD30 antigen) LIM domain only 6 RPRpSPPPRAP HLA-B7 49General transcription RPRpSPGSNSKV HLA-B7 50 factor 2-1Ajuba (a novel LIM protein GAQPGRHpSV HLA-B7 51required for mitotic commitment) novel retinal pigment SPRpSITSTP HLA-B752 epithelial cell protein/retinoic acid induced 14latent transforming growth KARpSPGRAL HLA-B7 53factor-beta-binding protein-2 No direct database hit- SPRpSPGRS(L/I)HLA-B7 54 (X = L/I) TGFB-induced factor LPRGSpSPSVL HLA-B7 55 2/TGIF2DNA-directed RNA FPHpSLLSVI HLA-B7 56 polymerase 1135 kDapolypeptide/POLR1B protein thyroid hormone receptor SPRERpSPAL HLA-B7 57associated protein 3 RhoGAP protein/Nadrin APRRYpSSSL HLA-B7 58Synemin/desmuslin RTFpSPTYGL HLA-B7 59 numb homolog SPFKRQLpSL HLA-B7 60(Drosophila)-like Chromatin assembly SPRSPpSTTYL HLA-B7 61factor 1, subunit A (p150) No direct database hit-RPApSP(K/Q)RA(K/Q)(L/I) HLA-B7 62 (X = L/I)-MIX?Interleukin enhancer-binding 62512150 KLFPDpTPLAL HLA-A2.1 63factor 3 (Nuclear factor of activated T-cells 90 kDa)Predicted: similar to RAVER1 55648233 RLLpSPLSSA HLA-A2.1 64MUM-2 (truncated) 20177848 RLDpSYVR HLA-A2.1 65 B lymphocyte signal 4261606 RQApSIELPSM HLA-A2.1 66 transduction gene KIAA1328 protein-20521886 KLMpSPKADVKL HLA-A2.1 67 hypothetical proteinSRp46 splicing factor 14141201 SMpTRSPPRV HLA-A2.1 68TFIID Transcription initiation  5032155 RLFpSKELRC HLA-A2.1 69 factor

In accordance with one embodiment of the present invention, a purifiedpolypeptide is provided comprising the amino acid sequence of SEQ ID NO:70-171, or an amino acid sequence that differs from any of thosesequences by one or more conservative amino acid substitutions. Inanother embodiment the purified polypeptide comprises an amino acidsequence that differs from SEQ ID NO: 70-171 by less than 5 conservativeamino acid substitutions, and in a further embodiment, by 2 or lessconservative amino acid substitutions. In accordance with one embodimentof the present invention, a purified polypeptide is provided comprisingthe amino acid sequence of SEQ ID NO: 70-171, or a bioactive fragment ofSEQ ID NO: 70-171, or an amino acid sequence that differs from SEQ IDNO: 70-171 by one to ten conservative amino acid substitutions. In oneaspect, a peptide of the invention is an unphosphorylated peptide havinga sequence identical with, or highly homologous with, one of thepeptides having SEQ ID NOs:70-171. The present invention is alsodirected to isolated nucleic acids, which comprise nucleic acidsequences encoding the non-phosphorylated homologs of thephosphopeptides of the invention. The polypeptides of the presentinvention may include additional amino acid sequences to assist in thestabilization and/or purification of recombinantly producedpolypeptides. These additional sequences may include intra- orinter-cellular targeting peptides or various peptide tags known to thoseskilled in the art. In one embodiment, the purified polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 70-171 and a peptide tag, wherein the peptide tag is linkedto the phosphorylated peptide sequence. Suitable expression vectors forexpressing such fusion proteins and suitable peptide tags are known tothose skilled in the art and commercially available. In one embodimentthe tag comprises a His tag. In another embodiment, the presentinvention is directed to a purified polypeptide that comprises an aminoacid fragment of a phosphorylated polypeptide.

More particularly the phosphorylated polypeptide fragment consists ofnatural or synthetic portions of a full-length polypeptide selected fromthe group consisting of SEQ ID NO: 70-171 that are capable of specificbinding to their natural ligand. Alternatively, the fragment maycomprise an antigenic fragment, including fragments of 10-30, 12-19,8-12 or 9 amino acids in length, of a polypeptide selected from thegroup consisting of SEQ ID NO: 70-171.

In accordance with one embodiment, a composition is provided forinducing an immune response against a cancer-associated phosphopeptideas described herein. In one embodiment, the composition consists of apeptide comprising a sequence selected from the group consisting of SEQID NO: 70-171, and antigenic fragments of those sequences. Thecompositions can be combined with a pharmaceutically acceptable carrieror adjuvant and administered to a mammalian species to induce an immuneresponse. The immune response can take the form of an antibody response,a T helper response, or a CTL response. The immune response may begenerated in vitro or in vivo.

In accordance with one embodiment, the peptides can be used to immunizea non-human recipient such as a mouse, rat, or goat for the productionof antibodies that specifically recognize the peptides. Antibodies topeptides may be generated using methods that are well known in the art.In one embodiment, recombinantly produced peptides, or fragments thereofare used to generate antibodies against the phosphorylated peptides.

In accordance with one embodiment, an antibody is provided which bindsto a polypeptide of the invention. In one aspect, the polypeptide isselected from the group consisting of SEQ ID NOs: 70-171. In oneembodiment the antibody is a monoclonal antibody. The antibodies may beused with or without modification, and may be labeled by joining them,either covalently or non-covalently, with a reporter molecule. Inaddition, the antibodies can be formulated with standard carriers andoptionally labeled to prepare therapeutic or diagnostic compositions.

Antibodies to peptides may be generated using methods that are wellknown in the art. For the production of antibodies, various hostanimals, including rabbits, mice, rats, goats and other mammals, can beimmunized by injection with a peptide. They may be conjugated to carrierproteins such as KLH or tetanus toxoid. Various adjuvants may be used toincrease the immunological response, depending on the host species, andincluding but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Methods of immunization to achieve a polyclonal antibodyresponse are well known in the art, as are the methods for generatinghybridomas and monoclonal antibodies.

For preparation of monoclonal antibodies, any technique, which providesfor the production of antibody molecules by continuous cell lines inculture may be used. For example, the hybridoma technique originallydeveloped by Kohler and Milstein (1975, Nature 256:495-497), as well asthe trioma technique, the human B-cell hybridoma technique (Kozbor etal., 1983, Immunology Today 4:72), and the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al., 1985, in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In anadditional embodiment of the invention, monoclonal antibodies can beproduced in germ-free animals utilizing recent technology(PCT/US90/02545). According to the invention, human antibodies may beused and can be obtained by using human hybridomas (Cote et al., 1983,Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human Bcells with EBV virus in vitro (Cole et al., 1985, in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). In fact,according to the invention, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takedaet al., 1985, Nature 314:452-454) by splicing the genes from a mouseantibody molecule specific for epitopes of TAG polypeptides togetherwith genes from a human antibody molecule of appropriate biologicalactivity can be used; such antibodies are within the scope of thisinvention.

Antibodies generated in accordance with the present invention mayinclude, but are not limited to, polyclonal, monoclonal, chimeric (i.e.,“humanized” antibodies), single chain (recombinant), Fab fragments, andfragments produced by a Fab expression library. These antibodies can beused as diagnostic agents for the diagnosis of conditions or diseasescharacterized by expression or overexpression of antigen peptides (suchas cancer), or in assays to monitor a patients responsiveness to ananti-cancer therapy. In one embodiment antibodies specific for one ormore of the antigen peptides are used as diagnostics for the detectionof the antigen peptides in cancer cells.

The antibodies or antibody fragments of the present invention can becombined with a carrier or diluent to form a composition. In oneembodiment, the carrier is a pharmaceutically acceptable carrier. Suchcarriers and diluents include sterile liquids such as water and oils,with or without the addition of a surfactant and other pharmaceuticallyand physiologically acceptable carrier, including adjuvants, excipientsor stabilizers. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose, and relatedsugar solution, and glycols such as, propylene glycol or polyethyleneglycol, are preferred liquid carriers, particularly for injectablesolutions.

In accordance with one embodiment, the detection of antigen peptides isused as a diagnostic mark for detecting cancer. In another embodimentthe antigen peptides can be used to immunize an individual to induce animmune response. The induced response may include T helper cells or CTLspecific for the antigen peptides. The induced immune response may beuseful in preventing the development of cancer in an individual withoutcancer, and it may be useful in eliminating or preventing the furtherspread of the disease in an individual with cancer. In one embodimentthe antigen peptides may be added to antigen presenting cells. Theantigen presenting cells will now present antigen peptides, which can beused to stimulate an in vitro T helper cell or CTL response. The Thelper cells or CTL can then be used as diagnostics to detect thepresence of tumor cells. The T helper cells or CTL can also be infusedinto a cancer patient as a treatment for cancer.

Accordingly, one embodiment of the invention is directed to the use ofantigen peptides as diagnostic markers for neoplastic disease such ascancer. The method comprises the steps of screening for elevated levelsor inappropriate expression of antigen peptides, including theexpression of antigen peptides in somatic tissues. The term“inappropriate expression” includes any non-typical expression that isdeleterious to the cell or host organism, including for example,expression in a cell type that normally does not express the peptide, orexpression of a modified form of the peptide that impacts cell function.Such screens could be conducted using antibodies specific for theantigen. Alternatively, antibodies directed against antigen peptides canbe used in assays to monitor patients being treated with anticancertherapies to monitor the effectiveness of the therapy.

The antigen peptides are known to be expressed in melanoma, ovarian,breast, colorectal, or squamous carcinoma of the lung, and thus may beused as immunogens to prevent, eliminate, or delay the progression of,inter alia, these types of cancer.

In one aspect, the cancer is, inter alia, sarcoma, renal cell carcinoma,pancreatic carcinomas, squamous tumors of the head and neck.

These same antigen peptides may also be expressed in untested forms ofcancer and thus may be useful in their ability to prevent, eliminate, ordelay the progression of additional cancers.

Antibodies generated with specificity for the antigen peptides are usedin accordance with one embodiment to detect the corresponding peptidesin biological samples. The biological sample could come from anindividual whom is suspected of having cancer and thus detection wouldserve to diagnose the cancer. Alternatively, the biological sample maycome from an individual known to have cancer, and detection of theantigen peptides would serve as an indicator of disease prognosis ortreatment efficacy. Appropriate immunoassays are well known in the artand include, but are not limited to, immunohistochemistry, flowcytometry, radioimmunoassay, western blotting, and ELISA. Biologicalsamples suitable for such testing would include, but are not limited to,cells, tissue biopsy specimens, whole blood, plasma, serum, sputum,cerebrospinal fluid, pleural fluid, and urine.

Antigens recognized by T cells, whether helper T lymphocytes or CTL, arenot recognized as intact proteins, but rather as small peptides thatassociate with class I or class II MHC proteins on the surface of cells.During the course of a naturally occurring immune response antigens thatare recognized in association with class II MHC molecules on antigenpresenting cells are acquired from outside the cell, internalized, andprocessed into small peptides that associate with the class II MHCmolecules. Conversely, the antigens that give rise to proteins that arerecognized in association with class I MHC molecules are generallyproteins made within the cells, and these antigens are processed andassociate with class I MHC molecules. It is now well known that thepeptides that associate with a given class I or class II MHC moleculeare characterized as having a common binding motif, and the bindingmotifs for a large number of different class I and II MHC molecules havebeen determined. It is also well known that synthetic peptides can bemade which correspond to the sequence of a given antigen and whichcontain the binding motif for a given class I or II MHC molecule. Thesepeptides can then be added to appropriate antigen presenting cells,either in vitro or in vivo, and be used to stimulate a T helper cell orCTL response. The binding motifs, methods for synthesizing the peptides,and methods for stimulating a T helper cell or CTL response are all wellknown and readily available.

The antigens of this invention may take the form of antigen peptidesadded to autologous dendritic cells and used to stimulate a T helpercell or CTL response in vitro. The in vitro generated T helper cells orCTL can then be infused into a patient with cancer (Yee et al., 2002),and specifically a patient with a form of cancer that expresses one ormore of antigen peptides. The antigen peptides may also be used tovaccinate an individual. The antigen peptides may be injected alone, butmost often they would be administered in combination with an adjuvant.The proteins may also be added to dendritic cells in vitro, with thedendritic cells being subsequently transferred into an individual withcancer with the intent of stimulating an immune response.

Peptide analogs can readily be synthesized that retain their ability tostimulate a particular immune response, but which also gain severalbeneficial features which include, but are not limited to the following:(i) Substitutions may be made in the peptide at residues known tointeract with the MHC molecule. Such substitutions can have the effectof increasing the binding affinity of the peptide for the MHC moleculeand can also increase the lifespan of the peptide-MHC complex, theconsequence of which is that the analog is a more potent stimulator ofan immune response than is the original peptide. (ii) The substitutionsmay be at positions in the peptide that interact with the receptor onthe T helper cells or CTL, and have the effect of increasing theaffinity of interaction such that a stronger immune response isgenerated. (iii) Additionally, the substitutions may have no effect onthe immunogenicity of the peptide per se, but rather than may prolongits biological half-life or prevent it from undergoing spontaneoussubstitutions or alternations which might otherwise negatively impact onthe immunogenicity of the peptide.

The antigen peptides of this invention can also be used as a vaccine forcancer, and more specifically for melanoma, ovarian, breast, colorectal,or lung squamous cancer. The antigens may take the form of genes,proteins, or peptides. The vaccine may include only the antigens of thisinvention or they may include other cancer antigens that have beenidentified.

Pharmaceutical carriers, diluents and excipients are generally addedthat are compatible with the active ingredients and acceptable forpharmaceutical use. Examples of such carriers include, but are notlimited to, water, saline solutions, dextrose, or glycerol. Combinationsof carriers may also be used. The vaccine compositions may furtherincorporate additional substances to stabilize pH, or to function asadjuvants, wetting agents, or emulsifying agents, which can serve toimprove the effectiveness of the vaccine.

The composition may be administered parenterally or orally, and, ifparenterally, either systemically or topically. Parenteral routesinclude subcutaneous, intravenous, intradermal, intramuscular,intraperitoneal, intranasal, transdermal, or buccal routes. One or moresuch routes may be employed. Parenteral administration can be, forexample, by bolus injection or by gradual perfusion over time.Alternatively, or concurrently, administration may be by the oral route.

It is understood that the suitable dosage of an immunogen of the presentinvention will depend upon the age, sex, health, and weight of therecipient, the kind of concurrent treatment, if any, the frequency oftreatment, and the nature of the effect desired, however, the mostpreferred dosage can be tailored to the individual subject, asdetermined by the researcher or clinician. The total dose required forany given treatment will commonly be determined with respect to astandard reference dose based on the experience of the researcher orclinician, such dose being administered either in a single treatment orin a series of doses, the success of which will depend on the productionof a desired immunological result (i.e., successful production of a Thelper cell and/or CTL-mediated response to the antigen, which responsegives rise to the prevention and/or treatment desired). Thus, theoverall administration schedule must be considered in determining thesuccess of a course of treatment and not whether a single dose, given inisolation, would or would not produce the desired immunologicallytherapeutic result or effect. Thus, the therapeutically effective amount(i.e., that producing the desired T helper cell and/or CTL-mediatedresponse) will depend on the antigenic composition of the vaccine used,the nature of the disease condition, the severity of the diseasecondition, the extent of any need to prevent such a condition where ithas not already been detected, the manner of administration dictated bythe situation requiring such administration, the weight and state ofhealth of the individual receiving such administration, and the soundjudgment of the clinician or researcher. Needless to say, the efficacyof administering additional doses, and of increasing or decreasing theinterval, may be re-evaluated on a continuing basis, in view of therecipient's immunocompetence (for example, the level of T helper celland/or CTL activity with respect to tumor-associated or tumor-specificantigens).

The concentration of the T helper or CTL stimulatory peptides of theinvention in pharmaceutical formulations are subject to wide variation,including anywhere from less than 0.01% by weight to as much as 50% ormore. Factors such as volume and viscosity of the resulting compositionshould also be considered. The solvents, or diluents, used for suchcompositions include water, possibly PBS (phosphate buffered saline), orsaline itself, or other possible carriers or excipients. The immunogensof the present invention may also be contained in artificially createdstructures such as liposomes, which structures may or may not containadditional molecules, such as proteins or polysaccharides, inserted inthe outer membranes of said structures and having the effect oftargeting the liposomes to particular areas of the body, or toparticular cells within a given organ or tissue. Such targetingmolecules may commonly be some type of immunoglobulin. Antibodies maywork particularly well for targeting the liposomes to tumor cells.

The present invention is also directed to a vaccine in which a peptideor polypeptide or active fragment of the present invention is deliveredor administered in the form of a polynucleotide coding the peptide orpolypeptide or active fragment, whereby the peptide or polypeptide oractive fragment is produced in vivo. The polynucleotide may be includedin a suitable expression vector and combined with a pharmaceuticallyacceptable carrier.

The vaccine compositions may be used prophylactically for the purposesof preventing cancer in an individual that does not currently havecancer, or they may be used to treat an individual that already hascancer. Prevention relates to a process of prophylaxis in which theindividual is immunized prior to the induction or onset of cancer. Forexample, individuals with a history of severe sunburn and at risk fordeveloping melanoma, might be immunized prior to the onset of thedisease. Alternatively, individuals that already have cancer can beimmunized with the antigens of the present invention so as to stimulatean immune response that would be reactive against the cancer. Aclinically relevant immune response would be one in which the cancerpartially or completely regresses and is eliminated from the patient,and it would also include those responses in which the progression ofthe cancer is blocked without being eliminated.

In one embodiment, the present invention provides methods of screeningfor agents, small molecules, or proteins that interact with polypeptidescomprising a sequence selected from the group consisting of SEQ ID NO: 1through 69 or bioactive fragments thereof. The invention encompassesboth in vivo and in vitro assays to screen small molecules, compounds,recombinant proteins, peptides, nucleic acids, antibodies, etc., whichbind to or modulate the activity of antigen peptides and are thus usefulas therapeutic or diagnostic markers for cancer.

As used herein, modulating the activity of an antigen peptide includesinterfering or altering the antigen peptide ligand binding properties.

EXAMPLES

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teachings providedherein.

Example 1

Phosphorylated peptides were extracted from melanoma cell lines thatexpress either or both of HLA-B7 and HLA-A*0201, identified by massspectrometry to be differentially displayed on melanoma versus a controlB cell line, and then sequenced. The peptides were identified throughthe following procedure. Two melanoma cell lines and one Blymphoblastoid cell line were extracted with detergent containingbuffer, and HLA-A*0201 class I MHC molecules were purified byimmunoaffinity chromatography. Peptides were separated from the MHCmolecules by extraction in acid and filtration through a 5000 daltoncut-off filter. Phosphopeptides were identified through analysis bymicrocapillary reversed phase high performance liquid chromatographytandem mass spectrometry. Sequences were determined from an analysis ofcollision activated dissociation spectra. Source proteins weredetermined from a search of protein and DNA databases. SEQ ID NOs:1through 69 were identified (see Table 1). One of ordinary skill in theart would appreciate that such techniques can be modified and that othertechniques are known to aid in identifying peptides of the invention.

These peptides represent potential targets of an immune response, eithercytotoxic T lymphocyte or antibody, that could be used for eithertherapeutic or diagnostic purposes. While the peptides have beenidentified on melanoma cells, they may also be expressed on other kindsof cancer cells and the invention covers their use for cancers otherthan melanoma. The binding of these peptides to class I MHC molecules isnecessary for their recognition by cytotoxic T lymphocytes, whilerecognition by antibody could occur in either a class I MHC associatedor free form. The invention comprises these peptides, together withstructural modifications that retain or enhance the ability: 1) to bindto MHC molecules or; 2 to stimulate an immune response or; 3 to berecognized by a product of an immune response.

The binding of the peptides to class I MHC molecules can be determinedby 2-3 anchor residues within the sequence, and these peptides aregenerally 8-11 residues in length. For example, peptides that bind toHLA-B7 generally contain a Pro at the second position, a hydrophobicaliphatic residue at the carboxyl terminus, and are 9 residues long.However, some peptides have been identified that do not contain one ofthese anchor residues, and peptides up to 14 residues in length havealso been identified. It has also been shown that other residues in asequence may augment or diminish binding, despite the presence orabsence of appropriate anchor residues.

The sequences displayed on a cell are derived from proteolysis ofproteins made inside the cell and transported into the lumen of theendoplasmic reticulum. The specificities of the proteases and thetransporter are poorly understood, and the sequences of all proteinsmade by human cells, based on data from the Human Genome Project, arevery incomplete. In addition, proteins may undergo modifications such asphosphorylation. However, relatively few of these sites have beenidentified. Thus, there is a large universe of potential peptidesdisplayed by any given MHC molecule based on the sequences of allproteins made by the cell and the distribution of appropriate anchormotifs within those sequences. However, the exact peptides displayed bya cell are not readily predictable. The direct identification of suchpeptides by mass spectrometry provides information that cannot beotherwise obtained at present without undue effort.

Different MHC molecules may have similar anchor preferences, leading tothe possibility that a peptide associated with, for example, HLA-A*0201,may also be displayed by HLA-A3. The existence of such “supertypes”means that the peptides identified above in association with one MHCmolecule may be presented by others, broadening their utility asantigens.

Example 2

CTL specific for two of the antigen peptides were generated by long termculture with the peptides. Two CTL lines specific for the antigenpeptide GLLGpSPVRA, lines 6850 and 6960, SEQ ID NO: 13, and two CTLlines specific for the antigen peptide RVApSPTSGV, SEQ ID NO: 14, lines5183 and 63 were used to detect these two antigen peptides on cancercells. The phosphopeptide-specific CTL (50,000 CTL/well) were incubated24 hours with the following cancer cell lines or EBV-transformed Blymphoblastoid cell lines (BLCL) (25,000 cells/well): COV413.AAD.A4ovarian carcinoma, DM331.AAD.A4 and SLM2.AAD.A1 melanomas, MCF7.AAD.A2and MDAMB231.AAD breast carcinomas, and JY EBVBLCL. Supernatants wereharvested 24 hours later and evaluated for the presence of murine IFN□(produced by murine CTL lines) by ELISA (eBioscience Ready-Set-go murineIFN□ ELISA kit). As a positive control, cancer cells were pulsed withthe specific antigen peptide (1 □M) to show that they are capable ofpresenting exogenously added peptide. In FIG. 1A, twophosphopeptide-specific CTL cell lines, 6850 and 6960, specific for thephosphopeptide GLLGpSPVRA, recognize the phosphopeptide on all thecancer cell lines, but not the control cell line. In FIG. 1B, twophosphopeptide-specific CTL cell lines, 5183 and 63, specific for thephosphopeptide RVApSPTSGV, recognize the phosphopeptide on all thecancer cell lines, but not the control cell line.

Example 3

Over-expression of select phospho-proteins is a hallmark of malignanttransformation.

Due to the low expression levels of phosphopeptides by the MHC class Ipathway, identification of immunologically relevant phospho-peptides isdifficult.

Secreted WIC molecule technology allows for an increase in the amountsof WIC class I peptides available for analysis.

Identification of WIC class I phosphopeptides that are unique to tumorcells provides an avenue to the production of peptide-based cancervaccines and biomarkers

Materials, Methods, and Results

Table 2 as set forth in FIG. 2 presents phosphopeptides of the presentinvention on breast cancer by the Class I WIC Molecule, in particular,on the HLA B*0702 allele. Table 3, as set forth below, summarizes“Source proteins for peptides identified from analyses of samples frombreast cancer and immortalized cell lines and their expression innon-tumorigenic control cell line 184B5 and the breast cancer cell linesBT20 and MCF-7”.

FIG. 3 graphically demonstrates

-   -   The MHC class I pathway—    -   a) Protein is marked for degradation    -   b) The proteasome digests the peptides into smaller fragments    -   c) Peptides are transported into the ER via the transporter        associated protein (TAP)    -   d) The MHC molecule with peptide loaded is transported to the        surface

FIG. 4 schematically illustrates “Sample analysis overview. Cells aregrown in culture and the cell supernatant is collected. MHC moleculesare immunoaffinity purified and peptides are eluted under acidicconditions. Peptides are esterified, enriched for phospho-peptidesthrough IMAC and/or TiO2 and analyzed via C18 micro electrospray LC/MSwith high resolution full MS and low resolution MS/MS”.

FIG. 5 describes the B*0702 motif showing amino acid anchors.

FIG. 6 shows the total number of peptides in IMAC and TiO2 experimentsand schematically depicts the overlap between the peptides identified inan IMAC analysis of 184B5 and a TiO2 analysis. The IMAC analysis wasperformed twice, once with ETD fragmentation (d.d. top 6) and once withCAD (d.d. top 10) fragmentation. The TiO2 analysis was performed in asingle run with toggled ETD/CAD fragmentation. We investigated each MSanalysis for the presence of all identified sequences using thecalculated accurate mass value.

The MHC class I pathway (FIG. 3 ) is classically responsible forpresenting endogenous peptides to the immune system. Each MHC moleculehas a binding pocket which can only accept peptides with narrowlydefined motifs. The MHC molecule with peptide loaded in the bindinggroove is transported to the surface where the MHC molecule remainsanchored to the membrane Phospho-peptides can be processed and displayedby the MHC class I pathway.

Secreted MHC Molecules

-   -   A cDNA clone of a specific MEW molecule is altered to remove the        regions that code for the transmembrane and cytoplasmic portions        of the protein    -   Cloned into a plasmid expression vector    -   Cell produce endogenous MHC molecules as well as the secreted        MEW molecules (sHLA)    -   sHLA are released into the cell culture medium as they lack the        portion of the protein responsible for anchoring them to the        membrane    -   The inventors have demonstrated that sHLA molecules successfully        present phospho-peptides and that they display a similar        repertoire of phospho-peptides as endogenous HLA molecules.

Phosphopeptide Analyses

-   -   Breast cancer cell lines (BT20, MCF7) and an immortalized        non-malignant mammary tissue cell line (184B5) were transfected        with the B*0702 sHLA cDNA1    -   FIG. 5 shows the motif for peptides which are presented by        B*0702 MHC molecules    -   Samples were analyzed with both ETD fragmentation and CAD        fragmentation on either an Orbitrap or FT-ICR instrument, which        were in-house custom modified for front end electron transfer        dissociation    -   Species of interest were identified by a combination of        algorithm searching (OMSSA), d0/d3 methyl ester pair detection        and CAD neutral loss detection    -   Peptide sequences were determined by manual confirmation of        search data and de novo sequencing utilizing both CAD and ETD        data where necessary    -   Source proteins were determined by BLAST2 search, and a single        protein identification was determined from UniProt 3

Due to issues with non-specific binding having a negative effect on theability to identify peptides in IMAC experiments, TiO2 was considered asan alternative. FIG. 4 shows the total number of peptides in IMAC andTiO2 experiments. The level of non-specific binding for non-phosphopeptides was reduced substantially in the TiO2 experiment, howeverrecovery of the angiotensin II phosphate (DRVyIHPF) standard peptide wasmuch lower in the TiO2 experiment.

Conclusions—Example 3

-   -   104 phospho-peptides have been identified, deriving from 84        different source proteins    -   22 phospho-peptides were only detected in samples deriving from        breast cancer cell lines    -   2 peptides were identified in both cancer cell lines but not in        the control cell line    -   Peptides from source proteins known to be involved in tumors        have been identified    -   In a complementary TiO2/IMAC experiment, 34 peptides were common        to both analyses, 14 peptides were unique to the IMAC experiment        and 35 peptides were unique to the TiO2 experiment

Based on the disclosure provided herein, the present inventionencompasses compositions and methods useful for submitting/testingspecific peptides of immunological interest for binding and T-cellrecognition assays. The present invention further encompassescompositions and methods to apply the sHLA technology to cell lines withknown phospho-peptide expression patterns and the present invention hasapplication to cells with non-ideal HLA alleles and/or cells with poorgrowth.

BIBLIOGRAPHY

-   1. Oriana E. Hawkins, Rodney S. VanGundy, Annette M. Eckerd,    Wilfried Bardet, Rico Buchli, Jon A. Weidanz, and William H.    Hildebrand, Journal of Proteome 10 Research, 2008, 7, 1445-1457.-   2. Altschul S F; Gish W; Miller W; Myers E W; Lipman D J, Journal of    Molecular Biology, 1990, 215, 403-410.-   3. The UniProt Consortium, The Universal Protein Resource (UniProt)    in 2010, Nucleic Acids Research, 2010, D142-D148 15

TABLE 3 Uniprot protein Uniprot identification 184B5 BT20 MCF7 proteinidentification 184B5 BT20 MCF7 A-kinase X X Unique peptide, X X X anchorprotein 13 unidentified source 6 Anky corbin X Unique peptide, Xunidentified source 7 Arginine- X Unique peptide, X X glutamic acidunidentified source 8 dipeptide repeats protein Ataxin-2 X X X Neuronnavigator 1 X Ataxin-2 - with X X Nuclear factor X point mutationerythroid 2-related factor 1 Ataxin-2-like X X Nuclear receptor X Xprotein coactivator 1 AT-rich X X Numb-like protein X X X interactivedomain- containing protein 1A Beta- X X X Partner and X adrenergiclocalizer of BRCA2 receptor kinase 1 Butyrate X X Phosphatidylinositol Xresponse factor 2 4,5-bisphosphate 5phosphatase A Cdc42- X XPlakophilin-3 X X interacting protein 4 Centrosomal X X Plakophilin-3 XX protein of 55 kDa Chondroitin X Plakophilin-3 X sulfate synthase 2Chromatin X X Pleckstrin X assembly homology domain- factor 1 subunit Acontaining family G Coiled-coil X X Pleckstrin X domain-containinghomology-like protein 6 domain family B member 1 Cullin-4A X XProline-rich protein 11 X Death-associated X proteasome non- X proteinkinase 1 ATPase regulatory subunit 4 DENN X Protein AF-17 X domain-containing protein 4C DNA X Protein ajuba X replication factor Cdt1DNA-binding X Protein TANC2 X X protein inhibitor ID-2 DNA-directed XPutative X RNA phosphoserine polymerase 1 phosphatase-like subunit RPA2protein Dynamin-1- X Rab11 family- X like protein interacting protein 1E3 ubiquitin- X Ras-associated and X X protein ligase pleckstrinhomology NEDD4-like X domains-containing X X protein 1 E3 ubiquitin-Ras-like protein X X protein ligase family member NEDD4-like 11A E3ubiquitin- Receptor X protein ligase expression- UBR5 enhancing protein4 Eukaryotic Receptor- X translation interacting initiation factorserine/threonine- 4B protein kinase 4 General RelA-associated Xtranscription inhibitor factor II-I Helicase X Retrotransposon- X XSKI2W derived protein PEG10 Histone-lysine Retrotransposon- XN-methyltransferase derived protein PEG10 SETD2 Homeobox X Rho GTPase- Xprotein TGIF2 activating protein 30 Hypoxia- Rho GTPase- X inducibleactivating protein factor 1-alpha 30 Krueppel-like RNA X factor 10pseudouridylate synthase domain Latent- Serine/arginine X transformingrepetitive matrix growth factor X protein 2 X beta-binding protein 2Leucine zipper X X X Serine/threonine- X X protein 1 protein kinase SIK1Lipolysis- X X X Serine/threonine- X X stimulated protein kinaselipoprotein tousled-like 1 receptor MAP7 domain- X X Signal-induced Xcontaining proliferation- protein 1 associated 1-like protein 1MICAL-like X Ski oncogene X protein 1 MICAL-like X X Splicing factor, XX X protein 2 arginine/serine-rich 7 MICAL-like X X Sterol regulatory XX protein 2 element-binding protein Microtubule- X Targeting protein Xactin cross- for Xklp2 linking factor 1 Mitogen-activated X X Tensin-4 Xprotein kinase kinase kinase 11 Mitogen- X Thyroid hormone X activatedreceptor-associated protein kinase protein 3 kinase kinase 11 Myocyte- XTranscription factor X specific HIVEP2 enhancer factor 2DMyocyte-specific X Transformer-2 X enhancer factor 2D protein homologbeta Myocyte- X Transient receptor X specific potential cation enhancerfactor 2D channel Myosin X X TSC22 domain X regulatory light familyprotein 4 chain 12A Unique X X Ubiquitin carboxyl- X peptide, terminalhydrolase unidentified 43 source 1 Unique X Uncharacterized X peptide,protein Clorf106 unidentified source 2 Unique X X Uncharacterized X Xpeptide, protein C6orf64 unidentified source 3 Unique X UncharacterizedX peptide, protein CXorf66 unidentified source 4 Unique XUncharacterized X peptide, protein KIAA0819 unidentified source 5 YTHdomain- X Uncharacterized X X containing protein KIAA1310 protein 1 Zincfinger X Yorkie homolog X protein 335 X X

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated by reference herein intheir entirety. One of skill in the art will appreciate that thesuperiority of the compositions and methods of the invention relative tothe compositions and methods of the prior art are unrelated to thephysiological accuracy of the theory explaining the superior results.

Headings are included herein for reference and to aid in locatingcertain sections. These headings are not intended to limit the scope ofthe concepts described 10 therein under, and these concepts may haveapplicability in other sections throughout the entire specification.

The previous description of the disclosed embodiments is provided toenable any person skilled in the art to make or use the presentinvention. Various modifications to these embodiments will be readilyapparent to those skilled in the art, and the generic principles definedherein may be applied to other embodiments without departing from thespirit or scope of the invention. The appended claims are intended to beconstrued to include all such embodiments and equivalent variations.Accordingly, the present invention is not intended to be limited to theembodiments shown herein but is to be accorded the widest scopeconsistent with the principles and novel features disclosed herein.

1. An isolated phosphopeptide of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20 amino acids comprising a sequence selected from the groupconsisting of SEQ ID NOs: 70-171 or a sequence at least 90% identicalthereto.
 2. The isolated phosphopeptide of claim 1, wherein saidsequence is selected from group consisting of SEQ ID NOs: 84, 87, 96,102, 118, 121, 125, 126, 128, 132, 138, 145, 155, 164, and 165 or asequence of at least 90% identical thereto.
 3. The isolatedphosphopeptide of claim 1, wherein said sequence is selected from thegroup consisting of SEQ ID NOs: 118, 121, 132, 145, 164, and 165 or asequence of at least 90% identical thereto.
 4. A composition comprisingone or more phosphopeptides of claim 1 and a pharmaceutically acceptablecarrier wherein the number is selected from the group consisting of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 and
 30. 5. The composition of claim 4,wherein said composition has the ability to stimulate a T cell mediatedimmune response to at least one of said phosphopeptides.
 6. Thecomposition of claim 4, comprising a peptide capable of binding to theHLA-B*0702 allele on the WIC class I molecule.
 7. The composition ofclaim 4, wherein the composition is capable of increasing the 5-yearsurvival rate of breast cancer patients treated with the compositionrelative to average 5-year survival rates that could have been expectedwithout treatment with the composition.
 8. The composition of claim 1,wherein said composition comprises one or more phosphopeptidescomprising a sequence selected from the group consisting of SEQ ID NOs:84, 87, 96, 102, 118, 121, 125, 126-, 128, 132, 138, 145, 155, 164, and165 and a pharmaceutical carrier.
 9. An in vitro population of dendriticcells comprising the composition of claim
 4. 10. An antibody orantibody-like molecule that specifically binds to a phosphopeptidecomprising the sequence selected from the group consisting of SEQ IDNOs: 70-171.
 11. The antibody or antibody-like molecule of claim 10,wherein the phosphopeptide comprises a sequence selected from the groupconsisting of SEQ ID NOs: 84, 87, 96, 102, 118, 121, 125, 126, 128, 132,138, 145, 155, 164, and
 165. 12. The antibody or antibody-like moleculeof claim 10, wherein the antibody or antibody-like molecule ispolyclonal, monoclonal, chimeric antibody, Fab fragments, or fragmentsproduced by a Fab expression library or a T-cell receptor.
 13. Acomposition comprising the antibody or antibody-like molecule of claim10, further comprising a pharmaceutically acceptable carrier.
 14. Thecomposition of claim 13, further comprising an adjuvant.
 15. A method ofinducing an immunogenic response comprising administering to a patientin need thereof a dose of the composition of claim
 4. 16. A method oftreating or preventing cancer comprising administering to a patient inneed thereof a dose of the composition of claim
 4. 17. A method oftreating or preventing breast cancer comprising administering to apatient in need thereof a dose of a composition comprising an isolatedphosphopeptide comprising a sequence selected from the group consistingof SEQ ID NOs:70-171 in combination with a pharmaceutically acceptablecarrier.
 18. The method of claim 17, wherein the isolated phosphopeptidecomprises a sequence selected from the group consisting of SEQ ID NO:84, 87, 96, 102, 118, 121, 125, 126, 128, 132, 138, 145, 155, 164, and165.
 19. A method of treating or preventing breast cancer comprisingadministering to a patient in need thereof the population of thedendritic cells of claim 9 in combination with a pharmaceuticallyacceptable carrier.
 20. A population of T-cells specific for aphosphopeptide of claim 1.